Process for the production of 10-hydroxy-19-nortestosterone by rhizopus



PROCESS FOR THE PRODUCTION OF 10- HYDROXY -19- NORTESTOSTERONE BYRHIZOPUS Herbert C. Murray, Hickory'Corners, and Durey H. Peterson,Kalamazoo Township, Kalamazoo County, Mich., assignors to The UpjohnCompany, Kalamazoo, Mich., a corporation of Michigan No Drawing.Application November 21, 1955 Serial No. 548,254

1 Claim. (Cl. 19551) The present invention relates to steroid compoundsand is more particularly concerned with 10-hydroxy-l9- nortestosterone(l0,17fi-dihydroxy-4-estren-3-one), the 17 3-esters thereof and theprocess of production therefore.

The new compounds of the present invention, the 10-hydroxy-19-nortestosterone and the 1713-esters thereof can berepresented by the following structural formula:

wherein R is selected from the group consisting of hydrogen and the acylradical of a hydrocarbon carboxylic acid containing from one to eightcarbon atoms, inclusive.

The lO-hydroxy-l9-nortest0sterone is prepared by exposing19-nortestosterone or any of the l7fi-esters of 19- nortestosterone tothe oxygenating action of a fungus of the order Mucorales preferably ofthe family Mucoraceae and specifically of the genus Rhizopus asdescribed in United States application Serial No. 180,496, filed August19, 1950, or Patent No. 2,602,769, and separating the thus produced10-hydroxy-l9-nortestosterone by chromatography and recrystallization.

This application is a continuation-in-part of our priorfiled copendingapplication Serial No. 297,242, filed July 5, 1952.

In the preparation of the esters of 10-hydroxy-l9 nortestosterone, thelO-hydroxy-19-nortestosterone is admixed with an acylatingagent such as,for example, ketene, a ketene of a selected organic carboxylic acid,acid containing preferably between one and eight carbon atoms,inclusive, an acid, acid chloride, or acid bromide, or an acidanhydride, or other known acylating agent, usually in a solvent such as,for example, pyridine or the like, or in an inert solvent, includingsolvents like benzene, toluene, ether, and the like, and heated at atemperature between about zero degrees centigrade and the boiling pointof the reaction mixture, usually about room temperature, for a periodbetween about one half hour and about 96 hours. The time of reaction issomewhat dependent upon the temperature at which the reaction is carriedout, the acylating agent, and the ratio of reactants. The reactionmixture is quenched with ice or cold water and the product is collectedin an organic solvent which is thereafter washed with a mildly basicsolution and water to obtain a solution of the product which isessentially neutral. In some instances, the product may be crystallizedfrom the reaction mixture, in which event it may be advantageous toseparate the product by filtration or other means, such as, for example,by recrystallization ed States Patent frame. suitable solvent or-bychromatographic purification, as deemed necessary.

It is an object of the present invention to provide10-hydroxy-19-nortestosterone and the 17B-organic carboxylic acidsthereof. It is a particular object of the present invention to provide aprocess for the production of lO-hydroxy-l9-nortestosterone and the l78-organic carboxylic acid acylates, preferably hydrocarbon carboxylicacid acylates wherein the acyl radical contains from one to eight carbonatoms, inclusive, of'l0-hydroxy-l9-nortestos terone. in the art to whichthis invention pertains.

The compounds of the present invention IO-hydroxy- 19-nortestosteroneand the esters thereof are useful as oral' or parenteral anabolicagents, having in addition estrogenic properties. TheIO-hydroxy-l9-nortestosterone and the esters thereof are also importantintermediates for the production of estradiol; in the presence of astrong acid, such as mineral acid, a simultaneous dehydration andrearrangement produces estradiol (cf. Example 11). In carrying out theprocess of the present invention 1- nortestosterone, suitably in asolvent is subjected to a growth of a species of the fungus of the genusRhizopus. Though Rhizopus nigricans is the preferred species otherspecies of the genus Rhizopus are useful in the instant process such as,for example, Rh. arrhizus, Rh. microsporus, Rh. speciosus, Rh. equinus,Rh. circinas, Rh. oligosporus, Rh. pusillus, Rh. bovinus, Rh. Cambodia,Rh. chinensz's, Rh. tritz'ci, Rh. suinus, Rh. oryzae, and the like.

The following examples illustrate the process and prod-.

nets of this invention, but the invention is not to be con strued aslimited thereto.

Example I .1 O-hydroxy-I 9-n0rmethyl testosterone A medium was preparedof twenty grams of corn steep liquor and ten grams of Cerelose orglucose diluted to one liter with water and adjusted to a pH between 4.8and 5. One hundred liters of the sterilized medium thus prepared wereinoculated with Rhizopus nigrz'cans, American Type- Culture CollectionNo. 6227b, and incubated for 24 hours at a temperature of 28 degreescentigrade using a rate of aeration of two liters of air per minute at200 rpm. agitation. After 24 hours of aeration and agitation a. solutionof 25 grams of nortestosterone, dissolved in 500 milliliters of acetone,was added. After an additional 24 hour period of incubation the beer andmycelium were extracted with acetone. The mycelium was filtered, washedtwice each time with a volume of acetone approximately equal to thevolume of the mycelium and extracted twice each time with a volume ofmethylene chloride approximately equal to the volume of the mycelium.The acetone and methylene chloride extract including solvent were addedto the beer filtrate. The mixed extract and beer filtrate were extractedsuccessively with two one-half by volume portions of methylene chlorideand with two one-fourth portions of methylene chloride. The combinedmethylene chloride extracts were washed with two one-tenth by volumeportions of a two percent aqueous solution of sodium bicarbonate andthen with two one-tenth by volume portions of water. After drying themethylene chloride extract with about three to five grams of anhydroussodium sulfate per liter of solution and filtering, the solvent wasremoved by distillation. The thus ob tained residue, 62 grams, weretreated as follows: Digestion of the extractives with milliliters ofpetroleum ether in four portions removed 34 grams of oil, leaving 28grams of insoluble residue containing practically all the steroid.Separation of the residue-was accomplished by exhaustive chromatographyover Florisil magnesium silicate as follows:

The 28 grams of petroleum ether-insoluble material was digested in 1.5liters of warm benzene for one hour.

Other objects will be apparent to those skilled 4.1 grams of crystals of6 3-hydroxy-19-nortestosterone was removed by filtration and thefiltrate was thereupon adsorbed on 800 grams of Florisil. The column waseluted withpetroleum ether containing progressively increasing amountsof acetone. 250 milliliter fractions were collected as follows:

Solvent Petroleum ether-acetone 97:3. Petroleum ether-acetone 96:4.Petroleum ether-acetone 92:8. Petroleum ether-acetone 90:10. Petroleumether-acetone 88:12. Petroleum etheracetone 87:13. Petroleumether-acetone 85:15.

257-292. Petroleum ether-acetone 80:20.

Fractions 131 to 190 were combined and after evaporation of the solventyielded 1.8 grams of crude 10,17t3-dihydroxy-4-estren-3-one. Thismaterial was recrystallized twice from ethyl acetate and methanol togive pure 10,17,8- dihydroxy-4-estren-3-onel-hydroxy-19-nortestosterone) of a melting point 199 to 205; rotation[zx] 76 degrees (C=O.594 percent, methanol) )3}; 237 m (15,025) 7553?3305, 1656, 1622 Cm.- (hydroxyl, carbonyl, and conjugated double bond).

Analysis.Calcd. for (3 1-1 0 C, 74.44; H, 9.03. Found: C, 74.45, 74.52;H, 9.21, 8.77.

Example 2.-hydroxy-Z9-nortesrosterone 1 7 3-acetate(10,17,6-dihyrlroxy-4-estren-3-0ne 1 7-acetate) A solution containingmilligrams of l0-hydroxy-l9- nortestosterone in 0.8 milliliter ofpyridine and 0.4 milliliter of acetic anhydride was allowed to stand fora period of 24 hours at room temperature (twenty to thirty degreescentigrade). The solution was thereafter poured into 35 milliliters ofwater and filtered to yield 36 milligrams of solids which wasrecrystallized from ethyl ether to give 19 milligrams oflO-hydroxy-19-nortestosterone 17-acetate of melting point 184 to 185degrees.

Analysis.-Calcd. for C H O C, 72.26; H, 8.49. Found: C, 72.81; H, 8.63.

Example 3.1O-hydroxy-I9-n0rtest0sterone 175-pr0pi0- naze In the samemanner as given in Example 2, lO-hydroxyl9-nortestosterone, dissolved inpyridine, is treated with propionic anhydride, to givel0-hydroxy-19-nortestosterone 17fi-propionate.

Example 4.10-hydroxy-Z 9-nortestoster0ne I 7B-bazyrate In the samemanner as given in Example 2, IO-hydroxyl9-nortestosterone, dissolved inpyridine, is treated with butyric anhydride, to giveIO-hydroxy-19-nortestosterone 1 7,6-butyrate.

Example 5.10-hydroxy-J9-n0rtest0ster0ne 1 7B-valerate hr the same manneras given in Example 2, 10-hydroxyl9-nortestosterone, dissolved inpyridine, is treated with valeric anhydride, to give10-hydroxy-19-nortestosterone l7fi-valerate.

Example 6 .--1 O-h ydroxy-I 9-nortestoster0ne 17B-benzoate In the samemanner as given in Example 2, IO-hydroxyl9-nortestosterone, dissolved inpyridine, is treated with benzoyl chloride, to givelO-hydroxy-l9-nortestosterone 1713-benzoate.

' 4 Example 7.10-hydroxy-1 9-nortestosterone 17,8-(fl-cyclopenlylpropionate) In the same manner as given in Example 2,l0-hydroxyl9-nortestosterone, dissolved in pyridine, is treated withfi-cyclopentylpropionyl bromide, to give lO-hydroxy-19- nortestosterone17p-(B-cyclopentylpropionate).

Example 8.-10-hydr0xy-19-n0rtest0ster0ne 17p-phenylacetate In the samemanner as given in Example 2, IO-hydroxy- 19-nortestosterone, dissolvedin pyridine, is treated with phenylacetyl chloride, to give10-hydroxy-19-nortestosterone l7B-phenylacetate.

Example 9.-10-hydroxy-I9-n0rtest0ster0ne 17,8-phenylpropionate In thesame manner as given in Example 2, lO-hydroxy- 19-nortestosterone,dissolved in pyridine, is treated with phenylacetyl bromide, to give10-hydroxy-19-nortestosterone l7fl-phenylpropionate.

Example 10.-10-hydr0xy-19-n0rtest0ster0ne 17,8-acylates acetate, acidquinolate, nicotinate, piperonate, 2-furoate,-

thioglycollate, para-chlorobenzoate, para-bromobenzoate, ortho-, meta-,para-nitrobenzoate, 3,5-dinitrobenzoate, benzenesulfonate,toluenesulfonate, para-nitrobenzenesulfonate,3,S-dinitrobenzenesulfonate, benzenephosphonate and the like.

Example 1 1 .Eslraa'i0l from 10-hydroxy-IQ-nortestos- 1 er one A mixtureof milligrams of 10-hydroxy-19- nortestostcroae, five milliliters ofacetic acid and one drop of sulfuric acid was heated on a steam bath fora period of one-half hour. The mixture was thereupon poured into fiftymilliliters of ice water and the precipitate collected on filter.Recrystallization of the precipitate from acetone and Skellysolve Bhexanes produced pure estradiol.

We claim:

A process for the production of 10-hydroxy-19 nortestosterone whichcomprises: growing a fungus of the genus Rhizopus under aerobicconditions, in the presence of a nutrient medium containing assimilablenonsteroidal,

carbon and 19-nortestosterone, and recovering the resulting10-hydroxy-19-nortestosterone.

References Cited in the file of this patent UNITED STATES PATENTS Murrayet a1 July 8, 1952 Murray et al July 13, 1954 Murray et al Oct. 19, 1954

